LAUSR

Site‐specific dynamics in proteins are at the heart of protein function. While electron paramagnetic resonance (EPR) has potential to measure dynamics in large protein complexes, the reliance on flexible nitroxide labels is limitating especially for the accurate measurement of site‐specific β‐sheet dynamics. Here, we employed EPR spectroscopy to measure site‐specific dynamics across the surface of a protein, GB1. Through the use of the double Histidine (dHis) motif, which enables labeling with a Cu(II) – nitrilotriacetic acid (NTA) complex, dynamics information was obtained for both α‐helical and β‐sheet sites. Spectral simulations of the resulting CW‐EPR report unique site‐specific fluctuations across the surface of GB1. Additionally, we performed molecular dynamics (MD) simulations to complement the EPR data. The dynamics observed from MD agree with the EPR results. Furthermore, we observe small changes in gǁ values for different sites, which may be due to small differences in coordination geometry and/or local electrostatics of the site. Taken together, this work expands the utility of Cu(II)NTA‐based EPR measurements to probe information beyond distance constraints.